Heteroatomic oligonucleoside linkages

ABSTRACT

Oligonucleotide-mimicking macromolecules that have improved nuclease resistance are provided. Replacement of the normal phosphorodiester inter-sugar linkages found in natural oligonucleotides with three or four atom linking groups provide unique oligonucleotide-mimicking macromolecules that are useful in regulating RNA expression and in therapeutics. Methods of synthesis and use also are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of application Ser. No.07/903,160, filed on Jun. 24, 1992, now abandoned, which is acontinuation-in-part of application PCT/US92/04294, filed on May 21,1992. Application PCT/US92/04294 is a continuation-in-part ofapplication Serial No. 07/703,619, filed on May 21, 1991 (now U.S. Pat.No. 5,378,825), which is a continuation-in-part of application SerialNo. 07/566,836, filed on Aug. 13, 1990 (now U.S. Pat. No. 5,223,618),and application Ser. No. 07/558,663, filed on Jul. 27, 1990 (now U.S.Pat. No. 5,138,045). Each of these patent applications is assigned tothe assignee of this patent application and are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates to the design, synthesis and application ofnuclease resistant macromolecules that function as oligonucleotidemimics and are useful for therapeutics, diagnostics and as researchreagents. The macromolecules have modified linkages in place of thephosphorodiester inter-sugar linkages found in wild type nucleic acids.The macromolecules are resistant to nuclease degradation and are capableof modulating the activity of DNA and RNA. Methods for synthesizing themacromolecules and for modulating the production of proteins, utilizingthe macromolecules of the invention are also provided. Further providedare intermediate compositions useful in the synthesis of themacromolecules.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are effected by proteins. Such proteins, actingeither directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man.

Classical therapeutics has generally focused upon interactions with suchproteins in an effort to moderate their disease causing or diseasepotentiating functions. Recently, however, attempts have been made tomoderate the actual production of such proteins by interactions with themolecules, i.e., intracellular RNA, that direct their synthesis. Theseinteractions have involved the hybridization of complementary"antisense" oligonucleotides or certain analogs thereof to RNA.Hybridization is the sequence-specific hydrogen bonding ofoligonucleotides or oligonucleotide analogs to RNA or single strandedDNA. By interfering with the production of proteins, it has been hopedto effect therapeutic results with maximum effect and minimal sideeffects. In the same way, oligonucleotide like macromolecules maymodulate the production of proteins by an organism.

The pharmacological activity of antisense oligonucleotides andoligonucleotide analogs, like other therapeutics, depends on a number offactors that influence the effective concentration of these agents atspecific intracellular targets. One important factor foroligonucleotides is their stability in the presence of nucleases. It isunlikely that unmodified oligonucleotides will be useful therapeuticagents because they are rapidly degraded by nucleases. Modifications ofoligonucleotides to render them resistant to nucleases is thereforegreatly desired.

Modifications of oligonucleotides to enhance nuclease resistance havegenerally taken place on the phosphorus atom of the sugar-phosphatebackbone. Phosphorothioates, methyl phosphonates, phosphoramidates andphosphorotriesters have been reported to confer various levels ofnuclease resistance. However, phosphate modified oligonucleotides havegenerally suffered from inferior hybridization properties. See Cohen, J.S., ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRCPress, Inc., Boca Raton Fla., 1989).

Another key factor is the ability of antisense compounds to traverse theplasma membrane of specific cells involved in the disease process.Cellular membranes consist of lipid-protein bilayers that are freelypermeable to small, non-ionic, lipophilic compounds and inherentlyimpermeable to most natural metabolites and therapeutic agents, seeWilson, D. B. Ann. Rev. Biochem. 47:933-965 (1978). The biological andantiviral effects of natural and modified oligonucleotides in culturedmammalian cells have been well documented. Thus, it appears that theseagents can penetrate membranes to reach their intracellular targets.Uptake of antisense compounds into a variety of mammalian cells,including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8cells has been studied using natural oligonucleotides and certainnuclease resistant analogs. For alkyl triester analogs, results havebeen reported by Miller, P. S., Braiterman, L. T. and Ts'O, P. O. P.,Biochemistry 16:1988-1996 (1977). For methyl phosphonate analogs,results have been reported by Marcus-Sekura, C. H., Woerner, A. M.,Shinozuka, K., Zon, G., and Quinman, G. V., Nuc. Acids Res. 15:5749-5763(1987); Miller, P. S., McFarland, K. B., Hayerman, K. and Ts'O, P. O.P., Biochemistry 16:1988-1996 (1977); and Loke, S. K., Stein, C., Zhang,X. H. Avigan, M., Cohen, J. and Neckers, L. M. Top. Microbiol. Immunol.141:282:289 (1988).

Often, modified oligonucleotides are less readily internalized thantheir natural counterparts. As a result, the activity of many previouslyavailable, modified antisense oligonucleotides has not been sufficientfor practical therapeutic, research or diagnostic purposes. Two otherserious deficiencies of prior modified oligonucleotides are inferiorhybridization to intracellular RNA and the lack of a defined chemical orenzyme-mediated event to terminate essential RNA functions.

Modifications to enhance the effectiveness of antisense oligonucleotidesand overcome these problems have taken many forms. These modificationsinclude modifications of the heterocyclic base, modifications of thesugar, and modifications of sugar-phosphate backbone. Priorsugar-phosphate backbone modifications, particularly on the phosphorusatom, have effected various levels of resistance to nucleases. Theability of an antisense oligonucleotide to bind to specific DNA or RNAwith fidelity is fundamental to antisense methodology. However, modifiedphosphorus oligonucleotides have generally suffered from inferiorhybridization properties. Replacement of the phosphorus atom has beenone approach to avoid the problems associated with modified phosphorousoligonucleotides. Certain modifications have been reported in whichreplacement of the phosphorus atom is effected. Matteucci, M.,Tetrahedron Letters 31:2385-2388 (1990), reported replacement of thephosphorus atom with a methylene group. However, such replacement islimited by the difficulties associated with uniform insertion of aformacetal linkage throughout an oligonucleotide backbone. Cormier, etal., Nucleic Acids Research 16:4583-4594 (1988) reported the replacementof the phosphorus moiety with a diisopropylsilyl moiety. Stirchak, etal., Journal of Organic Chemistry 52:4202-4206 (1987) reportedreplacement of the phosphorus linkage by short homopolymers containingcarbamate or morpholino linkages. Both of these replacements are limitedby a lack of suitable synthetic methodology and the low solubility andweak hybridization properties of the resultant molecules. Mazur, et al.,Tetrahedron 40:3949-3956 (1984) reported replacement of the phosphoruslinkage with a phosphonic linkage. This replacement has not beendeveloped beyond the synthesis of a homotrimer molecule. Goodchild, J.,Bioconjugate Chemistry 1:165-187 (1990) reported replacement by esterlinkages. However, ester linkages are enzymatically degraded byesterases and are therefore unsuitable as a replacement for thephosphate bond in antisense applications.

A recent publication by Tronchet, J. et. al, J. Carbohydrate Chemistry,10:723 (1991) reported the use of an oxyimino intergylcosidic linkagebetween two monosaccharides to form a disaccharide. In forming thislinkages, a first carbonyl sugar, either a hexose or a pentose, wasreacted with a second O-aminohexose sugar.

The limitations of the available methods for modification of thephosphorus backbone of oligonucleotides has led to a continuing and longfelt need for other modifications that might provide resistance tonucleases and satisfactory hybridization properties for antisenseoligonucleotide diagnostics, therapeutics, and research.

OBJECTS OF THE INVENTION

it is an object of the invention to provide macro-molecules thatfunction as oligonucleotide mimics for use in antisense oligonucleotidediagnostics, research reagents, and therapeutics.

It is a further object of the invention to provideoligonucleotide-mimicking macromolecules that possess enhanced cellularuptake.

Another object of the invention is to provide oligonucleotide-mimickingmacromolecules that have greater efficacy than unmodified antisenseoligonucleotides.

It is yet another object of the invention to provide methods forsynthesis and use of such oligonucleotide-mimicking macromolecules.

These and other objects shall become apparent to persons skilled in thearts to which this invention pertains given this specification and itsappended claims.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions that are useful formodulating the activity of an RNA or DNA molecule and that generallycomprise oligonucleotide-mimicking macromolecules. The macromoleculesare constructed from a plurality of linked nucleosides. In constructingthese macromolecules, the phosphorodiester linkage of the sugarphosphate backbone found in wild type nucleic acids has been replacedwith three and four atom linking groups. Such linking groups maintain adesired atomic spacing between the 3'-carbon of one nucleoside and the4'-carbon of an adjacent nucleoside. The oligonucleotide-mimickingmacromolecules of the invention comprise a selected linked sequence ofnucleosides that are specifically hybridizable with a preselectednucleotide sequence of single stranded or double stranded DNA or RNA.

The oligonucleotide-mimicking macromolecules of the invention aresynthesized conveniently, through solid state or solution methodology,to be complementary to or at least specifically hybridizable with apreselected nucleotide sequence of the RNA or DNA. Solid supportsynthesis is effected utilizing commercially available nucleic acidsynthesizers. The use of such synthesizers is generally understood bypersons of ordinary skill in the art as being effective in generatingnearly any desired oligonucleotide or oligonucleotide mimic ofreasonable length.

The oligonucleotide-mimicking macromolecules of the invention also caninclude nearly any modification known in the art to improve theproperties of wild type oligonucleotides. In particular, themacromolecules can incorporate modifications known to increase nucleaseresistance or hybridization.

In accordance with the present invention, novel macromolecules thatfunction as antisense oligonucleotide mimics are provided to enhancecellular uptake, nuclease resistance, and hybridization properties andto provide a defined chemical or enzymatically mediated event toterminate essential RNA functions.

It has been found that certain oligonucleotidemimicking macromoleculescan be useful in therapeutics and for other objects of this invention.At least a portion of the macromolecules of the invention has structure1: ##STR1## wherein one of L₁ or L₂ is O or S, and the other of L₁ or L₂is N--R; and combined L₃ and L₄ are CH₂, or L₃ is CH₂ and L₄ is CR'R";or

one of L₃ or L₄ is O or S, and the other of L₃ or L₄ is N--R; andcombined L₁ and L₂ are CH₂, or L₂ is CH₂ and L₁ is CR'R"; or

one of L₁ and L₄ is O, S or N--R, and the other of L₁ and L₄ is CR'R";and L₂ and L₃ are CH₂ ; or

L₁, L₂, L₃ and L₄, together, are O--N═CH--CH₂ or CH₂ --CH═N--O; or

L₁ is O; L₂ is N; L₃ is CH₂ ; and L₄ is C or CH; and together with atleast two additional carbon or hetero atoms, L₂, L₃ and L₄ form a 5 or 6membered ring; or

L₁ is C or CH; L₂ is CH₂ ; L₃ is N; and L₄ is O; and together with atleast two additional carbon or hetero atoms, L₁, L₂ and L₃ form a 5 or 6membered ring; and

R is H; C₁ to C₁₀ straight or branched chain lower alkyl or substitutedlower alkyl; C₂ to C₁₀ straight or branched chain lower alkenyl orsubstituted lower alkenyl; C₂ to C₁₀ straight or branched chain loweralkynyl or substituted lower alkynyl; a ¹⁴ C containing lower alkyl,lower alkenyl or lower alkynyl; C₇ to C₁₄ substituted or unsubstitutedalkaryl or aralkyl; a ¹⁴ C containing C₇ to C₁₄ alkaryl or aralkyl;alicyclic; heterocyclic; a reporter molecule; an RNA cleaving group; agroup for improving the pharmacokinetic properties of anoligonucleotide; or a group for improving the pharmacodynamic propertiesof an oligonucleotide;

R' and R" are H; or R' is H and R" is O--R; or combined R' and R" are═O;

X is H; O--R; S--R; NH--R; F, Cl; Br; CN; CF₃ ; OCF₃ ; OCN; SOCH₃ ; SO₂CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; a reporter molecule;an RNA cleaving group; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide;

Q is O or CH₂ ;

n is an integer greater than 0; and

Bx is a variable heterocyclic base moiety.

The remainder of the molecule is composed of chemical functional groupsthat do not hinder, and preferably enhance, hybridization with RNA orsingle stranded or double stranded DNA.

In certain preferred embodiments, the macromolecules of structure 1include macromolecules wherein one of L₁ or L₂ is O or S, and the otherof L₁ or L₂ is N--R; and combined L₃ and L₄ are CH₂ ; or one of L₃ or L₄is O or S, and the other of L₃ or L₄ is N--R; and combined L₁ and L₂ areCH₂.

Other preferred embodiments of the invention include macromolecules ofstructure 1 wherein one of L₁ and L₄ is O, S or N--R, and the other ofL₁ and L₄ is CR'R'; and L₂ and L₃ are CH₂.

Further preferred embodiments of the invention include macromolecules ofstructure 1 wherein one of L₁ or L₂ is O or S, and the other of L₁ or L₂is N--R; and L₃ is CH₂ and L₄ is CR'R"; or one of L₃ or L₄ is O or S,and the other of L₃ or L₄ is N--R; and L₂ is CH₂ and L₁ is CR'R".

Further preferred embodiments of the invention include macromolecules ofstructure 1 wherein L₁, L₂, L₃ and L₄, together, are O--N═CH--CH₂ or CH₂--CH═N--O.

Further preferred embodiments of the invention include macromolecules ofstructure 1 wherein L₁ is O; L₂ is N; L₃ is CH₂ ; and L₄ is C or CH; andtogether with at least two additional carbon or hetero atoms, L₂, L₃ andL₄ form a 5 or 6 membered ring; or L₁ is C or CH; L₂ is CH₂ ; L₃ is N;and L₄ is O; and together with at least two additional carbon or heteroatoms, L₁, L₂ and L₃ form a 5 or 6 membered ring.

In particularly preferred embodiments of the invention, Q of structure 1is O. In accordance with other particularly preferred embodiments of theinvention, X of structure 1 is H or OH. In accordance with otherpreferred embodiments of the invention the Bx group of individualnucleosides incorporated within structure 1 are independently selectedfrom naturally occurring or synthetic purine and pyrimidine heterocyclicbases. Such heterocyclic bases include but are not limited to adenine,guanine, cytosine, thymine, uracil, 5-methylcytosine, hypoxanthine or2-aminoadenine. Other such heterocyclic bases include 2-methylpurine,2,6-diaminopurine, 6-mercaptopurine, 2,6-dimercaptopurine,2-amino-6-mercaptopurine, 5-methylcytosine,4-amino-2-mercaptopyrimidine, 2,4-dimercaptopyrimidine and5-fluorocytosine.

Particularly preferred embodiments of the invention includemacromolecules of structure 1 wherein L₁ is O or S, L₂ is N, L₃ is CH₂and L₄ is CH₂ or CHOR, particularly where R is H. In accordance withother particularly preferred embodiments of the invention, L₄ is O or S,L₃ is N, L₂ is CH₂ and L₁ is CH₂ or CHOR, particularly where R is H. Inaccordance with even further particularly preferred embodiments of theinvention, L₂ and L₃ both are CH₂ and one of L₁ or L₄ is N--R and theother is CH₂.

In preferred embodiments of the inventions, theoligonucleotide-mimicking macromolecules include from about 2 to about50 nucleoside subunits (i.e., n=about 1-about 49).

The oligonucleotide-mimicking macromolecules of the invention preferablyare included in a pharmaceutically acceptable carrier for therapeuticadministration.

The substituent groups of the above referenced alkyl, alkenyl, alkynyl,alkaryl and aralkyl R groups include but are not necessary limited tohalogen, hydroxyl, keto, carboxy, nitrates, nitrites, nitro, nitroso,nitrile, trifluoromethyl, O-alkyl, S-alkyl, NH-alkyl, amino, azido,sulfoxide, sulfone, sulfide, silyl, intercalators, conjugates,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligonucleotides, and groupsthat enhance the pharmacokinetic properties of oligonucleotides. Oneparticularly preferred R group is CF₃. Typical intercalators andconjugates include cholesterols, phospholipids, biotin, phenanthroline,phenazine, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Halogens include fluorine, chlorine,bromine, and iodine. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improveoligonucleotide uptake, enhance oligonucleotide resistance todegradation, and/or strengthen sequence-specific hybridization with RNA.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve oligonucleotide uptake,distribution, metabolism or excretion.

The invention further includes methods of modulating the production oractivity of a protein in a cell system or an organism comprisingcontacting the cell system or organism with an oligonucleotide-mimickingmacromolecule having structure 1.

The invention further includes methods of treating an organism having adisease characterized by the undesired production of a proteincomprising contacting the organism with an oligonucleotide-mimickingmacromolecule having structure 1.

The invention further includes methods of in vitro assaying asequence-specific nucleic acid comprising contacting a test solutioncontaining the nucleic acid with an oligonucleotide-mimickingmacromolecule having structure 1.

The invention further includes nucleosidic precursors of themacromolecules of structure 1, the precursors having structure 2:##STR2## wherein Y₁ is O; Y₂ is H or R'"; and Z is aminooxy orphthalimidooxy; or

Y₁ is CH₂, and Y₂ is aminooxy, alkylamino, aminooxyalkyl, alkenyl oroxoalkyl; and

Z is H, OH, O--R'", amino methyleneamino or phthalimido;

R'" is a hydroxyl blocking group;

X is H or OH;

Q is CH₂ or O; and

Bx is a heterocyclic base moiety.

In certain preferred embodiments, Y₁ is CH₂ ; Y₂ is aminooxy,alkylamino, hydroxyalkyl, aminooxyalkyl, alkenyl or aldoalkyl; and Z isH, OH or O--R'". In other preferred embodiments, Y₁ is O, Y₂ is H and Zis aminoxy or phthalimido.

DETAILED DESCRIPTION OF THE INVENTION

The term "nucleoside" refers to a unit composed of a heterocyclic baseand a sugar, generally a pentose sugar. In naturally occurringnucleosides, the heterocyclic base typically is guanine, adenine,cytosine, thymine or uracil. In naturally occurring nucleosides, thesugar is normally deoxyribose, i.e., erythro-pentofuranosyl, or ribose,i.e., ribopentofuranosyl. Synthetic sugars also are known, includingarabino, xylo or lyxo pentofuranosyl sugars and hexose sugars.Throughout this specification, reference to the sugar portion of anucleoside or other nucleic acid apecies shall be understood to refer toeither a true sugar or to a species replacing the traditional sugarmoiety of wild type nucleic acids. Additionally, reference to theheterocyclic base portion of a nucleoside or other nucleic acid speciesshall be understood to refer to either a natural, modified or syntheticbase replacing one or more of the traditional base moiety of wild typenucleic acids. Moreover, reference to inter-sugar linkages shall betaken to include moieties serving to join the sugar or sugar substitutemoiety together in the fashion of wild type nucleic acids.

The term "nucleotide" refers to a nucleoside having a phosphate groupesterified to one of its 2', 3' or 5' sugar hydroxyl groups. Thephosphate group normally is a monophosphate, a diphosphate ortriphosphate.

The term "oligonucleotide" refers to a plurality of monophosphatenucleotide units that typically are formed in a specific sequence fromnaturally occurring bases and pentofuranosyl sugars joined by nativephosphodiester bonds. A homo-oligonucleotide is formed from nucleotideunits having the same heterocyclic base, i.e. poly(A). The termoligonucleotide generally refers to both naturally occurring andsynthetic species formed from naturally occurring subunits.

The term "oligonucleotide analog" has been used in various publishedpatent application specifications and other literature to refer tomolecular species similarly to oligonucleotides but that havenon-naturally occurring portions. This term has been used to identifyoligonucleotide-like molecules that have altered sugar moieties, alteredbase moieties or altered inter-sugar linkages. Thus, the terminologyoligonucleotide analog has been used to denote structures having alteredinter-sugar linkages including phosphorothioate, methyl phosphonate,phosphotriester or phosphoramidate inter-nucleoside linkages used inplace of phosphodiester inter-nucleoside linkages; purine and pyrimidineheterocyclic bases other than guanine, adenine, cytosine, thymine oruracil and sugars having other than the β pentofuranosyl configurationor sugars having substituent groups at their 2' position orsubstitutions for one or more of the hydrogen atoms. The term "modifiedoligonucleotide" also has been used in the literature to denote suchstructures.

"Oligonucleotide mimics" as the term is used in connection with thisinvention, refers to macromolecular moieties that function similarly toor "mimic" the function of oligonucleotides but, have non-naturallyoccurring intersugar linkages. Oligonucleotide mimics thus can havenatural or altered or non-naturally occurring sugar moieties and naturalor altered or non-naturally occurring base moieties in combination withnon-naturally occurring inter-sugar linkages.

For the purposes of this invention, an oligonucleotide mimic havingnon-phosphodiester bonds, i.e. an altered inter-sugar linkage, canalternately be considered an "oligonucleoside" or an"oligonucleotide-mimicking macromolecule". The terms oligonucleoside oroligonucleotide-mimicking macromolecule thus refers to a plurality ofjoined nucleoside units connected by non-phosphate containing linkinggroups.

Additionally, the term "oligomers" is intended to encompassoligonucleotides, oligonucleotide analogs, oligonucleosides oroligonucleotide-mimicking macromolecules. Thus, in speaking of"oligomers" reference is made to a series of nucleosides or nucleosideanalogs that are joined together via either natural phosphodiester bondsor via other linkages, including the linkages of this invention.Generally, the linkage is from the 3' carbon of one nucleoside to the 5'carbon of a second nucleoside. However, the term "oligomer" can alsoinclude other linkages such as a 2'→5' linkage or a 3'→4' linkage.

Antisense therapy is the use of oligonucleotides or other oligomers forthe purpose of binding with complementary strands of RNA or DNA. Afterbinding, the oligonucleotide and the RNA or DNA strand can be consideredto be "duplexed" together in a manner analogous to native, doublestranded DNA. The oligonucleotide strand and the RNA or DNA strand canbe considered to be complementary strands in the same context as nativedouble stranded DNA. In such complementary strands, the individualstrands are positioned with respect to one another to allow Watson/Cricktype hybridization of the heterocyclic bases of one strand to theheterocyclic bases of the opposing strand.

Antisense therapeutics can be practiced in a plethora of organismsranging from unicellular prokaryotes and eukaryotes to multicellulareukaryotes. Any organism that utilizes DNA-RNA transcription orRNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to antisense therapeuticsand/or prophylactics. Seemingly diverse organisms such as bacteria,yeast, protozoa, algae, all plant and all higher animal forms, includingwarm-blooded animals, can be treated by antisense therapy. Further,since each of the cells of multicellular eukaryotes includes bothDNA-RNA transcription and RNA-protein translation as an integral part oftheir cellular activity, antisense therapeutics and/or diagnostics canalso be practiced on such cellular populations. Furthermore, many of theorganelles, e.g. mitochondria and chloroplasts, of eukaryotic cellsinclude transcription and translation mechanisms. Thus, single cells,cellular populations or organelles can also be included within thedefinition of organisms that are capable of being treated with antisensetherapeutics or diagnostics. As used herein, therapeutics is meant toinclude the eradication of a disease state, killing of an organism, e.g.bacterial, protozoan or other infection, or control of erratic orharmful cellular growth or expression.

Prior antisense therapy utilizing "oligonucleotide analogs" isexemplified in the disclosures of the following United States and PCTpatent applications: Ser. No. 463,358, filed Jan. 11, 1990, entitledCompositions And Methods For Detecting And Modulating RNA Activity; Ser.No. 566,836, filed Aug. 13, 1990, entitled Novel Nucleoside Analogs;Ser. No. 566,977, filed Aug. 13, 1990, entitled Sugar ModifiedOligonucleotides That Detect And Modulate Gene Expression; Ser. No.558,663, filed Jul. 27, 1990, entitled Novel Polyamine ConjugatedOligonucleotides;, Ser. No. 558,806, filed Jul. 27, 1991, entitledNuclease Resistant Pyrimidine Modified Oligonucleotides That Detect AndModulate Gene Expression; Ser. No. 703,619, filed May 21, 1991, entitledBackbone Modified Oligonucleotide Analogs; serial number PCT/US91/00243,filed Jan. 11, 1991, entitled Compositions and Methods For Detecting AndModulating RNA Activity; and patent application PCT/US91/01822, filedMar. 19, 1991, entitled Reagents and Methods For Modulating GeneExpression Through RNA Mimicry; all assigned to the assignee of thisinvention. The disclosures of each of the above noted patentapplications are herein incorporated by reference.

As is set forth in detail in the above-referenced United States and PCTpatent applications, oligonucleotides and other oligomers haveapplication in diagnostics, therapeutics, and as research reagents andkits. For therapeutic use, oligonucleotides or other oligomers would beadministered to an animal, including humans, suffering from a diseasestate that is desirous to treat.

This invention is directed to certain macromolecules that function likeoligonucleotides yet exhibit other useful properties. As is illustratedin the Examples and Schemes of this specification, the macromoleculesare constructed from basic nucleoside units. These nucleoside units arejoined by a linkage of the invention to form dimeric units. The dimericunits can be further extended to trimeric, tetrameric and other, higherorder macromolecules by the addition of further nucleosides. The dimericunits (and/or the higher order units) can be linked via linkages otherthan those of the invention, as for instance, via a normalphosphodiester linkage, a phosphorothioate linkage, a phosphoramidatelinkage, a phosphotriester linkage, a methyl or other alkylphosphonatelinkage, a phosphorodithioate linkage or other linkage.

In certain embodiments, a single linkage is used to join nucleosides toform a macromolecule of the invention. For example, in Scheme XVIIIbelow, m and r are 0, q is 1, and n and p are greater than 1. In otherembodiments, two or more different linkages are used. For example, inScheme XVIII, m and r are 0, q is 1, and n and p are greater than 1.

In other macromolecules of the invention the nucleoside are joinedtogether in groups of two, three or more nucleoside that together form aunit. An activated phosphityl moiety is located at the 3' terminus ofthis unit and a hydroxyl moiety bearing a removable hydroxyl blockinggroup is located at the 5' terminus. On subsequent removal of thehydroxyl blocking group and reaction of the hydroxyl group with anactivated phosphityl group, the units are then joined together via anormal phosphodiester, phosphorothioate or other phosphorus linkage.Thus a first unit (a group of two, three or more nucleosides linkedtogether via a first linkage of the invention) and to a second unit (agroup of two, three or more nucleosides linked together via the firstlinkage or via a second linkage of the invention) are connected togetherthrough a phosphate linkage. The macromolecule is elongated by theaddition of further units of nucleosides (linked together via the first,a second or additional linkages of the invention) by joining theseadditional units to the existing linked units via further phosphoruslinkages. In the examples and flow schemes shown below, unitsexemplified by compound 58 could be linked together or they could belinked to units of compounds 66, 72, 77, 81 or 85 or variouscombinations of these compounds could be linked together in variousmacromolecule structures. As is exemplified in Scheme XVIII below, insuch macromolecules r is 0 or 1, m is a positive number, q is greaterthan 1, and n and p are positive numbers.

Scheme I illustrates certain abbreviations used for blocking groups inother of the Schemes. Scheme I further shows the synthesis of 3'-O-aminoand 3'-O-methyleneamino nucleosides via a Mitsunobu reaction utilizingN-hydroxylphthalimide and methylhydrazine to generate an --O--NH₂ moietyon a sugar hydroxyl. The --O--NH₂ group can then be derivatized to a--O-methyleneamino moiety. These reactions are exemplified in Examples1, 2, 3, 5 and 15.

The reactions of Examples 1, 2, 3 and 5 represent an improved synthesisof 3'-O--NH₂ nucleosides. In forming --O--NH₂ moieties on sugars, it istheoretically possible to displace a leaving group, as for instance atosyl group, with hydroxylamine. However, Files, L. E., Winn, D. T.,Sweger, R. W., Johnson, M. P., and Czarnik, J. Am. Chem. Soc., 14:1493(1992) have shown that such a displacement leads to a preponderance of--NHOH moieties and not to the desired --O--NH₂ moieties. Further, thereaction sequence of Examples 1, 2, 3 and 5 represents an improvedsynthesis compared to that illustrated in European Patent Application 0381 335. The synthetic pathway of that patent application requires theuse of a xylo nucleoside as the staring material. Xylo nucleosides areless readily obtainable than the ribonucleoside utilized in Examples 1,2, 3 and 5.

Scheme II illustrates the conversion of a 4'-aldo nucleoside to a5'-aldo nucleoside. This reaction is exemplified in Example 16. SchemeIII illustrates the generation of a 5'-aldo methyl sugar. This isexemplified in Example 14. Scheme IV illustrates the formation of an5'-iodo nucleoside, exemplified in Example 6. Similar methodology isused to generate an active iodo group on a terminal hydroxyl of adimeric unit in Scheme X and Example 10. In Scheme IV, the iodonucleoside is further derivatized to a 6'-aldo nucleoside via an allosubstituted nucleoside. This is exemplified in Examples 31 and 32.

Scheme V illustrates a free radical reaction of a --O-methyleneaminonucleoside of Scheme 1 to a 5'-amino 5'-homo nucleoside. This isexemplified in Example 30. Scheme VI illustrates use of a Mitsunobureaction on a 5'-homo nucleoside to synthesize an oxyamine homonucleoside, i.e. a 6'-O--NH₂ 5'-homo nucleoside. This is exemplified inExamples 36, 37 and 38. Scheme VII illustrates N-alkylation of the aminomoiety of a 6'-amino-5'-deoxy'5-homo nucleoside. This is exemplified inExamples 43, 44 and 45. Such N-alkylation is desirable where the aminomoiety subsequently will be reacted with a thiol moiety. The N-alkylatedproduct of such a reaction exhibits greater stability to acid than doesthe non-alkylated S--N bond. This is particularly useful in solidsupport synthesis wherein acid removal of trityl groups is commonlypracticed. However, for other synthesis, such as solution synthesis,this may not be a concern.

Schemes VIII to XVII illustrate the use of the nucleosides of Schemes Ito VII for the assembly of dimeric, trimeric and other, higher orderoligonucleosides. In Scheme VIII, nucleosides 3 and 31 are joined via anacid catalyzed coupling reaction to form an --O-nitrilomethylidynelinkage between the respective two nucleosides. This is exemplified inExample 17. Dimeric oligonucleoside 32 can be reduced to animinomethylene linkage that, in turn, can be alkylated to a(methylimino)methylene linkage, as exemplified in Example 18.

Scheme IX illustrates the joining of nucleoside 3 to nucleoside 5. Thisscheme is analogous to Scheme VIII with the exception that in Scheme IXa three atom linkage is created whereas in Scheme VIII a four atomlinkage is created. Nucleosides 3 and 5 are joined in Step 1 to form an--O-nitrilo linkage that is reduced in Step 2 to an --O-imino linkage.Alkylation occurs in Step 3 to a --O-methylimino linkage, with finaldeblocking in Step 4. These steps are exemplified in Example 4. Thealkylation reaction in Step 3 is accompanied by deblocking thet-butyldimethylsilyl protecting group at the 5' terminus of the dimer.Advantageous use of this deblocking reaction also is utilized in otherSchemes. Deblocking of the t-butyldiphenylsilyl group used to protectthe 3' terminus of the dimer is effected using tetra-n-butylammoniumfluoride.

The alkylation step can be used to introduce other, useful, functionalmolecules on the macromolecule. Such useful functional molecules includebut are not limited to reporter molecules, RNA cleaving groups, groupsfor improving the pharmacokinetic properties of an oligonucleotide, andgroups for improving the pharmacodynamic properties of anoligonucleotide. Such molecules can be attached to or conjugated to themacromolecule via attachment to the nitrogen atom in the backbonelinkage. Alternatively, such molecules can be attached to pendent groupsextending from the 2' position of the sugar moiety of one or more of thenucleosides of the marcromolecules. Examples of such other usefulfunctional groups are provided by U.S. patent application Ser. No.782,374, filed Oct. 24, 1991, entitled Derivatized OligonucleotidesHaving Improved Uptake & Other Properties, assigned to the same assigneeas this application, herein incorporated by reference, and in other ofthe above-referenced patent .applications.

Scheme X illustrates a synthetic scheme utilized to prepare dimers,trimers, and other, higher order oligonucleosides having homogenouslinkages between nucleosides. In this scheme, nucleosides 10 and 12 arelinked to form an iminomethylene linkage as exemplified in Example 7.Advantageous use of the alkylating-5' terminus deblocking step of SchemeIX is effected to remove the blocking group at the 5' terminus of thedimeric oligonucleoside 14, as in Example 8. Using the iodinationreaction of Scheme IV, the dimer is then converted to a 5' terminus iodointermediate, as in Example 10. A further 3'-O-methyleneaminonucleosidic unit 10 then can be added to the dimer to form a trimer, asin Example 11, followed by deblocking and alkylation, as in Example 12.This reaction sequence can be repeated any number of times to form ahigher order oligonucleoside. The oligonucleoside is deblocked at the 3'terminus, as is exemplified for the dimer in Example 9 or the tetramerin Example 13.

Scheme XI illustrates the use of an 1-O-alkyl sugar that is first linkedto a nucleoside. Reduction followed by alkylation and deblocking yieldsan --O-(methylimino)methylene linkage joining the 1-O-alkyl sugar andthe nucleoside, as exemplified by Example 19. This structure is thenblocked at the 5' terminus, as exemplified by Example 20. The fullyblocked, linked sugar-nucleoside structure is then subjected toglycosylation to add a heterocyclic base to the sugar moiety and thusform a dimeric nucleoside structure, as in Example 21. Afterglycosylation, removal of the 5' terminus blocking group andchromatographic separation of α and β anomers, as exemplified by Example22, yields a dimer. This dimer can be further elongated as per theprocedure of Scheme X. Examples 23, 34 and 25 exemplify the addition ofan adenine, cytosine and guanine base to a thymidine-methyl sugar dimerto form T-A, T-C and T-G dimers in addition to the T-T dimer of SchemeX. Examples 26, 27 and 28 exemplify the formation of A-T, A-A, A-C, A-G,C-T, C-A, C-C, C-G, G-T, G-A, G-C and G-G dimers. Each may be furtherelongated as per the procedures of Scheme X.

Scheme XII illustrates a radical reaction that forms a linkage having apendant hydroxyl moiety. This is exemplified in Example 33. The pendantOH group can be oxidized to an ═O using Moffatt oxidization conditions.Alternatively, the pendant OH moiety can be cyclized to the nitrogenatom of the linkage to form either a five or a six membered heterocyclicring. The formation of a linkage incorporating a six atom ring isexemplified in Example 34. A five atom ring would be formed utilizingcondition analogous to those of Neumeyer, J. L. & Boyce, C. B., J. Org.Chem., 38:2291 (1973) to add phosgene in the presence of a base such astriethylamine or diethylphenylamine in toluene at a temperature of about60° to about 80° C.

Scheme XIII illustrates the formation of an iminooxymethylene linkage.Example 35 describes the preparation of the 5'-O-trityl protected xylostarting nucleoside and Example 39 describes the reaction of compound 50with compound. 54 to form a dimeric unit. Continuing within Scheme XIII,to prepare dimeric units that can be used as solid support buildingblocks (Example 40), the backbone nitrogen atom is alkylated, followedby simultaneous removal of both the 5'-O-trityl and the3'-O-(t-butyldiphenylsilyl) protecting groups with trifluoroacetic acid.The 5'-terminus hydroxyl group is blocked with dimethoxytriryl (Example41), followed by forming an active phosphoramidate dimer (Example 42).

Scheme XIV illustrates the preparation of a thiol 2 intermediate and theuse of that intermediate with an amino nucleoside to form aS-iminomethylene linkage (Example 45). As with the reactions of SchemeXIII, a dimeric unit having an active phosphoramidate moiety can beformed. This is exemplified by Examples 46 and 47.

Scheme XV illustrates the preparation of a nucleoside intermediate andcoupling of that intermediate to a further nucleoside, as exemplified inExample 48, to form a nitrilo-1,2-ethanediyl linkage. This linkage canbe reduced to an imino-1,2-ethanediyl linkage, as exemplified in Example49. Further, in a manner similar to Schemes XIII and XIV, Scheme XVillustrates the preparation of an active phosphoramidate species, asexemplified in Examples 50, 51 and 52.

Scheme XVI illustrates the preparation of a 2' substituted nucleoside,as exemplified in Example 53, and conversion of that 2' substitutednucleoside to a further nucleoside having an active linkage formingmoiety (Example 54). Linkage of this 2' substituted nucleoside to afurther nucleoside (Example 55) is followed by conversion to an activephosphoramidate (Example 56). Substitution of the 2' position in amacromolecule of the invention, as noted above, is useful for theintroduction of other molecules, including the introduction of reportermolecules, RNA cleaving groups, groups for improving the pharmacokineticproperties of an oligonucleotide, and groups for improving thepharmacodynamic properties of an oligonucleotide as well as other groupsincluding but not limited to O, S and NH alkyl, aralkyl, aryl,heteroaryl, alkenyl, alkynyl and ¹⁴ C containing derivatives of thesegroups, F, Cl, Br, CN, CF₃, OCF₃, OCN, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃,NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino and substituted silyl.

Further illustrated in Scheme XVI is the preparation of a carbocyclicnucleoside (Example 57), joining of that carbocyclic nucleoside with afurther nucleoside via a linkage of the invention (Example 58), andformation of an active phosphoramidate (Example 63). A further sequenceof reactions are also illustrated in Scheme XVI, wherein a carbocyclicnucleoside is derivatized at its 2' positions (Example 60) and convertedto a further nucleoside (Example 61). As with the other reactions ofthis scheme, a dimer is first formed (Example 62), and then derivatizedwith an active phosphoramidate (Example 63). The dimers of this schemehaving a 3' phosphoramidite moiety are used as in schemes XIII, XIV andXV to link the oligonucleosides of the invention to other nucleosidesvia a phosphodiester, phosphorothioate or other similar phosphate basedlinkage.

Scheme XVII illustrates a further carbocyclic containing, dimericnucleoside. Internucleoside conversion is exemplified in Examples 64 and65, and formation of a dimeric structure is exemplified in Example 66.The dimeric structure of Scheme XVII shows a carbocyclic nucleoside asthe 5' nucleoside of the dimer, while Scheme XVI shows a carbocyclicnucleoside as the 3' nucleoside of the dimer. Use of carbocyclicnucleosides for both nucleoside intermediates, in the manner asdescribed for other of the reaction schemes, results in a dimer having acarbocyclic nucleoside at both the 3' and 5' locations.

Scheme XVIII illustrates generic structures that are prepared from thenucleosides and oligonucleoside of the previous schemes. Exemplarymacromolecules of the invention are described for both solid support andsolution phase synthesis in Example 68. ##STR3##

EXAMPLE 1 5'-O-(t-Butyldimethylsilyl)-3'-O-Phthalimidothymidine, 2

To a solution of 5'-O-t-butyldimethylsilylthymidine [1, 21.36 g, 60mmol, prepared according to the procedure of Nair, V., and Buenger, G.S., Org. Prep. Procedures Int., 22:57 (1990) in dry THF (750 ml)],triphenylphosphine (17.28 g, 66 mmol) and N-hydroxyphthalimide (10.74 g,66 mmol) were added. The solution was cooled to 0° C. anddiisopropylazodicarboxylate (15.15 g, 75 mmol) was added dropwise over aperiod of 3 hr while stirring under nitrogen. The reaction mixture wasthen stirred at room temperature for 12 hr. The solution was evaporatedand the residue was dissolved in CH₂ Cl₂ (750 ml), extracted with sat.NaHCO₃ (200 ml), and water (200 ml), dried (MgSO₄), filtered andconcentrated to furnish yellow oily residue. Silica gel columnchromatography (100% hexanes, and then hexanes:Et₂ O gradient to 90% Et₂O) of the residue gave compound 2 as a colorless glass (18.68 g, 62%); ¹H NMR (CDCl₃) δ0.05 [2s, 6, (CH₃)₂ ], 0.91 [s, 9, (CH₃)₃ ], 2.0 (s, 3,CH₃), 2.5-2.65 (m, 2, 2'CH₂), 4.05-4.2 (m, 2, 5'CH₂), 4.25-4.35 (m, 1,4'H), 5.0 (m, 1, 3'H), 6.15 (m, 1, 1'H), 8.6 (br s, 1, NH), and aromaticprotons.

Anal. Calcd. for C₂₄ H₃₁ N₃ O₇ Si: C, 57.46; H, 6.23; N, 8.37. found :C, 57.20; H, 6.26; N, 8.27.

EXAMPLE 2 3'-O-Amino-5'-O-(t-Butyldimethylsilyl)thymidine, 3

Cold methylhydrazine (1.6 ml, 30 mmol) was added to a stirred solutionof 5'-O-(t-butyldimethylsilyl)-3'-O-phthalimidothymidine (2, 4.6 g, 9.18mmol) in dry CH₂ Cl₂ (60 ml) at 5°-10° C. After 10 minutes whiteprecipitation of 1,2-dihydro-4-hydroxy-2-methyl-1-oxophthalizineoccurred. The suspension was stirred at room temperature for 1 h. Thesuspension was filtered and precipitate washed with CH₂ Cl₂ (2×20 ml).The combined filtrates were concentrated and the residue purified bysilica gel column chromatography. Elution with CH₂ Cl₂ :MeOH(100:0→97:3, v/v) furnished the title compound (3.40 g, 100%) as whitesolid. Crystallization from CH₂ Cl₂ gave white needles, m.p. 171° C.; ¹H NMR (CDCl₃) δ0.05 [s, 6, (CH₃)₂ ], 0.90 [s, 9, (CH₃)₃ ], 2.22-2.58(2m, 2, 2'CH₂), 3.9-4.08 (m, 3, 5'CH₂, and 3'H) 4.30 (m, 1, 4H) 5.5 (brs, 2, NH₂) 6.2 (m, 1, 1'H) 7.45 (s, 1, C₆ H) 8.9 (br s, 1, NH).

Anal. Calcd. for C₁₆ H₂₉ N₃ O₅ Si: C, 51.72; H, 7.87; N, 11.32. found:C, 51.87, H, 7.81; N, 11.32.

EXAMPLE 3 3'-O-Aminothymidine, 4

3'-O-Amino-(t-butyldimethylsilyl)thymidine was deblocked with (Bu)₄NF/THF in standard way to furnish compound 4 (72%). Crystallized fromether/hexanes/ethanol as fine needles, mp 81° C. ¹ H NMR (Me₂ SO-d₆)δ1.78 (s, 3, CH₃), 2.17 and 2.45 (2m, 2, 2'CH₂), 3.70 (m, 2, 5'CH₂),3.88 (m, 1, 4'H), 4.16 (m, 1, 3'H), 4.8 (br s, 1, 5'OH), 6.05 (dd, 1,1'HM), 6.2 (br s, 2 NH₂), 7.48 (s, 1, C₆ H), and 11.24 (br s, 1, NH).

Anal. Calcd. for C₁₀ H₁₅ N₃ O₅ : C, 46.69; H, 5.87; N, 16.33; found: C,46.55; H, 5.91; N, 16.21.

EXAMPLE 43'-O-Dephosphinico-3'-O-(Methylimino)thymidylyl-(3'→5')-5'-Deoxythymidine,9

Step 1.

3'-O-Amino-5'-O-(t-butyldimethylsilyl)thyroidinc (3, 1.85 g, 5 mmol),3'-O-(t-butyldimethylsilyl)thyroidinc-5'-aldehyde [5, 2.39 g, 5 mmol;freshly prepared by following the method of M. J. Camarasa, F. G. De lasHeras, and M. J. Perez-Perez, Nucleosides and Nucleotides, 9:533 (1990)]and AcOH (0.25 ml) were stirred together in CH₂ Cl₂ (50 ml) solution atroom temperature for 2 h. The products were then concentrated underreduced pressure to give the intermediate oxime linked dimer, compound6.

Step 2.

The residue obtained from Step 1 was dissolved in AcOH (25 ml). NaCNBH₃(1.55 g, 25 mmol, in 3-portions) was added to the stirred AcOH solutionat room temperature. The solution was stirred for 30 min to give theintermediate imine linked dimer, compound 7.

Step 3.

Aqueous HCHO (20%, 2 ml 66 mmol) and additional NaCNBH₃ (1.55 g, 25mmol, in 3-portions) was added to the stirred reaction mixture of Step 2at room temperature. After 2 h, the solution was diluted with EtOH (100ml) , and resulting suspension was evaporated under reduced pressure.The residue was dissolved in CH₂ Cl₂ (150 ml) and then washedsuccessively with 0.1M HCl (100 ml), saturated aqueous NaHCO₃ (100 ml),and water (2×50 ml). The dried (MgSO₄) CH₂ Cl₂ solution was evaporatedto give crude methylated imine linked dimer 8.

Step 4.

The residue from Step 3 was dissolved in the THF (30 ml) and a solutionof (Bu)₄ NF (1M in THF, 10 ml) was added while stirring at roomtemperature. After 1 h, the reaction mixture was evaporated underreduced pressure and the residue was purified by short columnchromatography. The appropriate fractions, which eluted with CH₂ Cl₂:MeOH (8:2, v/v) were pooled and evaporated to give compound 9 as a foam(0.74 g, 30%). ¹ H NMR (Me₂ SO-d₆) δ1.78 (s, 6, 2CH₃), 2.10 (m, 4,2'CH₂), 2.5 (s, 3, N--CH₃), 2.8 (m, 2, 5'-N--CH₂), 3.6-4.08 (5m, 6, 5'CH₂, 4' CH, 3' CH), 4.75 and 5.3 (2 br s, 2, 3' and 5' OH), 6.02 (d, 1,1'H), 6.1 (t, 1, 1'H), 7.4 and 7.45 (2s, 2, 2C₆ H), 11.3 (br s, 2, NH).

EXAMPLE 55'-O-(t-Butyldimethylsilyl)-3'-Deoxy-3'-[(Methyleneamino)oxy]thymidine,10

A solution of HCHO (20% aqueous, 1 ml) was added dropwise to a stirredsolution of 3'-O-amino-5'-O-(t-butyldimethylsilyl)thymidine (3, 7.42 g,20 mmol) in dry MeOH (400 ml) at room temperature. After 6 h, anotherportion of HCHO (20% aqueous, 1.5 ml) was added and stirring continuedfor 16 h. The resulting solution was evaporated under reduced pressure,and the residue was purified by chromatography on silica gel to givecompound 10 (7.25 g, 95%) as clear foam. ¹ H NMR (CDCl₃) δ0.1 [s, 3,(CH₃)₂ ], 0.9 [s, 9, (CH₃)₃ ], 1.9 (s, 3, CH₃), 2.25-2.72 (m, 2, 2'CH₂), 3.85-4.15 (2m, 3, 5' CH₂, 4'H), 4.85 (m, 1, 3'H), 6.25 (dd, 1,1'H), 6.5 and 6.95 (2d, 2, N═CH₂), 7.43 (s, 1, (6H), 9.2 (br s, 1 NH).

EXAMPLE 6 3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Iodothymidine 12

To a stirred solution of 3'-O-(t-butyldiphenylsilyl)thymidine [11, 10.0g, 20.83 mmol, prepared according to the procedure of Koster, H. andSinha, N. D., Tet. Letts., 26:2641 (1982)] in dry DMF (375 ml) was addedmethyltriphenoxyphosphonium iodide (12.12 g, 30 mmol) under argon atroom temperature. The solution was stirred for 16 h. The DMF was removedunder reduced pressure and the residue was dissolved in CH₂ Cl₂ (500ml). The organic layer was washed with 20% aqueous Na₂ S₂ O₃ (200 ml),water (2×200 ml) and dried (MgSO₄). The solvent was evaporated and theresidue was purified by silica gel chromatography. Elution with Et₂ O:Hexanes (1:1, v/v), pooling of appropriate fractions and concentrationfurnished compound 12 as white power (7.87 g, 64%, mp 142° C.). Anal.Calcd. for C₂₆ H₃₁ N₂ O₄ SiI: C, 52.88; H, 5.29; N, 4.74; I, 21.33.Found: C,52.86; H, 5.21; N, 4.66; I, 21.54.

EXAMPLE 75'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,13

A stirred solution of5'-O-(t-butyldimethylsilyl)-3'-deoxy-3'-[(methyleneamino)oxy]thymidine(10, 1.62 g, 4.23 mmol),3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iodothymidine (12, 2.5 g, 4.23mmol), bis(trimethylstannyl)benzopinacolate [4.84 g, 8.46 mmol, preparedaccording to the method of Hillgartner, H; Neumann, W. P.; Schroeder,B., Liebigs Ann. Chem., 586-599 (1975)] in dry benzene (9 ml) wascarefully degassed 3-times (flushed with argon) and heated at 80° C. for8 h. The reaction mixture was cooled and concentrated under reducedpressure and the residue was purified by silica gel chromatography. Theappropriate fractions, which were eluted with CH₂ Cl₂ :MeOH (97:3, v/v),were pooled and concentrated to give dimeric oligonucleoside, compound13 (1.25 g, 35%) as white foam. ¹ H NMR (CDCl₃) δ0.09 and 0.13 [2s, 6,(CH₃)₂ ], 0.89 and 1.06 [2s, 9, (CH₃)₃ ], 1.07 and 1.08 [2s, 9, (CH₃)₃], 1.87, and 1.90 (2s, 6, 2 CH₃), 5.74 (br s, 1, NH), 6.20-6.31 (2m, 2,2 1'H), 6.88 (s, 1, C₆ H), 10.33 and 10.36 (2 br s, 2, 2NH) and otherprotons.

EXAMPLE 83'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method A

Compound 13 was treated as per the procedure of Step 3 of Example 4 tosimultaneously N-alkylate the imino nitrogen and deblock the 5' silylblocking group of the 5' nucleoside of the dimer to yield compound 14 asa foam. ¹ H NMR (CDCl₃) δ1.07 (s, 9, (CH₃)₃), 1.85 and 1.88 (2s, 6,2CH₃), 2.56 (s, 3, N--CH₃), 4.77 (br s, 1, 5' OH), 6.1 and 6.2 (2m, 2,1'H), 7.4 and 7.62 (2m, 10, Ph H), 9.05 (br s, 2, 2 NH), and otherprotons.

EXAMPLE 93'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-5'-Deoxythymidine,15

The 3'-O-(t-butyldiphenylsilyl) blocking group of compound 14 is removedas per the procedure of Step 4 of Example 4 to yield the fully deblockeddimeric oligonucleoside, compound 15.

EXAMPLE 103'-O-Dephosphinico-3'-O-[(Methylimino)methylene]-5'-Iodo5'-Deoxythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,16

Compound 14 is treated as per the procedure of Example 6 to yield thetitle dimeric oligonucleoside, compound 16, having a reactive iodofunctionality at the terminal 5' position and a blocking group remainingat the 3' position.

EXAMPLE 115'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deoxythymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,17

Compound 16 is reacted with compound 10 utilizing the conditions ofExample 7 to extend the oligonucleoside to yield the trimericoligonucleoside, compound 17.

EXAMPLE 123'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deoxythymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythyroidine,18

Compound 17 when reacted as per the conditions of Example 8 will undergoN-alkylation to the trimeric oligonucleoside and will be deblock at the5' position to yield compound 18, wherein n=2.

EXAMPLE 133'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deoxythymidylyl-(3'.fwdarw.5')-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]-5'-Deoxythymidylyl-(3'→5')-5'-Deoxythymidine,20

The sequence of Examples 10, 11 and 12 is repeated for the addition of afurther nucleoside to extend the oligonucleoside to a tetramer, compound19. The tetrameric oligonucleoside 19 is then treated as per theprocedure of Example 9 to remove the terminal 3' silyl blocking groupyielding the fully deblocked tetrameric oligonucleoside, compound 20.

EXAMPLE 14Methyl3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-5-C-Formyl-α/β-D-erythro-Pentofuranoside,23

2-Deoxy-D-ribose, 21, was modified to methyl2-deoxy-a/β-D-erythro-pentofuranoside (prepared according to the methodof M. S. Motawai and E. B. Pedersen, Liebigs Ann. Chem. 1990, 599-602),which on selective tosylation followed by 3-O-silylation gave methyl3-O-(t-butyldimethylsilyl)-2-deoxy-5-O-tosyl-α/β-D-erythro-pentofuranosidein overall 70% yield. The latter compound on iodination followed bycyanation gave the corresponding 5-C-cyano intermediate compound 22, asa syrup. ¹ H NMR (CDCl₃) δ1.05 (s, 9, (CH₃)₃) , 1.9-2.38 (m, 4, 2 CH₂),3.3 and 3.4 (2s, 3, OCH₃), 3.98-4.30 (3m, 2, 3, 4-CH), 4.95 and 5.05(2m, 1, 1H), 7.4 and 7.7 (2m, 10, Ph H) . IR (neat) 2253 cm⁻¹ (CH₂ CN)].Compound 22 (stored at 0° C. without any degradation) wasreduced(DIBAL-H) freshly every time as and when the title compound 23was required.

EXAMPLE 155'-O-(t-Butyldimethylsily1)-2',3'-Dideoxy-3'[(Methyleneamino)oxy]adenosine,27;5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-[(Methyleneamino)oxy]cytidine,28; and5'-O-(t-Butyldimethylsilyl)-2',3'-Dideoxy-3'-[(Methyleneamino)oxy]guanosine,29

3'-O-Amino-2'-deoxyadenosine, compound 24, 3'-O-amino-2'-deoxycytidine,compound 25, and 3'-O-amino-2'-deoxyguanosine, compound 26, prepared asper the procedures of European Patent Application 0 381 335 or in amanner analogous to the preparation of compound 4 by the procedure ofExample 3 above, are blocked at their 5' position with at-butyldimethylsilyl group according to the procedure of Nair, V., andBuenger, G. S., Org. Prep. Procedures Int., 22:57 (1990) tO give thecorresponding 3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyadenosine,3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxycytidine and3'-O-amino-5'-(t-butyldimethylsilyl)-2'-deoxyguanosine nucleosideintermediates. Treatment of the blocked intermediate as per theprocedure of Example 5 or as per the procedure of Preparation example 4of European Patent Application 0 381 335 gives the corresponding5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]adenosine,compound 27;5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]cytidine,compound 28; and5'-O-(t-butyldimethylsilyl)-2',3'-dideoxy-3'-[(methyleneamino)oxy]guanosine,compound 29.

EXAMPLE 16 3'-O-(t-Butyldiphenylsilyl)thymidine-6'-Aldehyde, 31

The title compound is prepared by homologation of the above described3'-O-(t-butyldimethylsilyl)thymidine-5'-aldehyde (compound 5) utilizingthe procedure of Barton, D. H. R. et al., Tet. Letts., 30:4969 (1989).The 5'-aidehyde, compound 5, is treated via a Witig reaction with(methoxymethylidene)triphenylphosphate. The resulting enol ether,compound 30, is hydrolyzed with Hg(OAc)₂, KI, H₂ O and THF according tothe procedure of Nicolaou, K. C., et al., J. Am. Chem. Soc., 102:1404(1980) to furnish the compound 31.

EXAMPLE 175'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Nitrilomethylidyne)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,32

The title compound is prepared by reaction of compound 31 and compound 3in the manner of Example 4, Step 1 to furnish the dimericoligonucleoside having an oxime backbone.

EXAMPLE 183'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method B

Compound 32 when treated as per the procedure of Steps 2 and 3 ofExample 4 will also yield compound 14.

EXAMPLE 19 Methyl3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]thymidylyl-(3'→5)-3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,33

Compound 23 and compound 3 are linked utilizing the procedure of Example4, Steps 1 to couple the sugar and the nucleoside via an oxime linkage.The resulting oxime linkage is then reduced utilizing the procedure ofExample 4, Step 2 to an iminomethylene linkage and this linkage, inturn, when N-alkylated via the procedure of Example 4, Step 3 will yieldcompound 33.

EXAMPLE 20 Acetyl5'-O-Benzoyl-3'-O-Dephosphinico-3'-O-[(Methyimino)methylene]thymidylyl-(3'→5)-3-O-(t-Butyldiphenylsilyl)-2,5-Dideoxy-α/β-D-erythro-Pentofuranoside,34

Compound 33 will be treated with benzoyl chloride according to theprocedure of Jenkins et al., Synthetic Procedures in Nucleic AcidChemistry, Zorbach and Tipson, Ed., Vol. 1, John Wiley & Sons, Pg. 149,to benzoylate the free 5'-hydroxyl of compound 33 which is hydrolyzedand acylated in situ according to the procedure of Baud et. al, Tet.Letts., 31:4437 (1990) to yield compound 34.

EXAMPLE 215'-Benzoyl-3'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,35

Compound 34 is reacted with silylated thymine as per the procedure ofBaud, et al., Tetrahedron Letters, 31:4437 (1990) utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene to yield5'-O-benzoyl-3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxythymidine,compound 35 as an anomeric mixture.

EXAMPLE 223'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,14

Method C

Compound 35 when treated with methanolic ammonia will also yieldcompound 14. Further treatment as per the procedure of Example 9 willyield the fully deblocked dimer, from which anomerically pure compound15 will be isolated by chromatography.

EXAMPLE 233'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyadenosine,36

Compound 34 is reacted with silylated adenine as per the procedure ofBaud, et al., Tetrahedron Letters, 31:4437 (1990) utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyadenosine,36.

EXAMPLE 243'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxycytidine37

Compound 34 is reacted with silylated cytosine as per the procedure ofBaud, et al., Tetrahedron Letters, 31:4437 (1990) utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxycytidine,37.

EXAMPLE 253'-O-Dephosphinico-3'-O-[(Methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxyguanosine38

Compound 34 is reacted with silylated guanine as per the procedure ofBaud, et al., Tetrahedron Letters, 31:4437 (1990) utilizingdibenzo-18-crown-6 and potassium iodide in acetonitrile-toluene. Removalof the benzoyl group with methanolic ammonia and chromatographicseparation will yield3'-O-dephosphinico-3'-O-[(methylimino)methylene]thymidylyl-(3'→5')-3'-O-(t-butyldiphenylsilyl)-5'-deoxyguanosine,38.

EXAMPLE 26 A-(3'→5')-T; A-(3'→5')-A; A-(3→'5')-C; and A-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoadenosine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is adenine. The linkednucleosidesugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the A-T, A-A, A-C and A-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis adenine and Bxj is thymine, adenine, cytosine and guanine,respectively.

EXAMPLE 27 C-(3'→5')-T; C-(3'→5')-A; C-(3'→5')-C; and C-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl).-3'-O-aminocytidine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is cytidine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the C-T, C-A, C-C and C-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis cytosine and Bxj is thymine, adeninc, cytosine and guanine,respectively.

EXAMPLE 28 G-(3'→5')-T; G-(3'→5')-A; G-(3'→5')-C; and G-(3'→5')-G3'-Dephosphinico-3'-(Methylimino)methylene Linked Dimers

In a manner analogous to the procedures of Examples 19 and 20, the5'-(t-butyldimethylsilyl)-3'-O-aminoguanosine intermediate of Example 15will be reacted with compound 3 to yield a linked nucleoside-sugarcompound equivalent to compound 34 wherein Bxi is guanine. The linkednucleoside-sugar intermediate will then be reacted as per the proceduresof Examples 21, 23, 24 and 25 to yield the G-T, G-A, G-C and G-G dimers,respectively, of a structure equivalent to that of compound 14 where Bxiis guanine and Bxj is thymine, adeninc, cytosine and guanine,respectively.

EXAMPLE 29 Trimeric, Tetrameric, Pentameric, Hexameric And Other HigherOrder Oligonucleosides Having a Selected Nucleoside Sequence

The dimers of Examples 21, 23, 24, 25, 26, 27 and 28 are extended byreaction with the5'-(t-butyldimethylsilyl)-3'-deoxy-3'-[(methyleneamino)oxy] nucleosides,compounds 10, 27, 28 and 29, of Examples 5 and 15 to form trimersutilizing the looping sequence of reactions of Examples 10, 11 and 12.Iteration of this reaction sequence loop adds a further nucleoside tothe growing oligonucleoside per each iteration of the reaction sequenceloop. The reaction sequence loop of Examples 10, 11 and 12 is repeated"n" number of times to extend the oligonucleoside to the desired "n+1"length. The final 3'-blocked oligonucleoside when treated as per theprocedure of Example 9 to remove the terminal3'-O-(t-butyldiphenylsilyl) blocking group will yield the fullydeblocked oligonucleoside of the selected nucleoside sequence andlength.

EXAMPLE 30 6'-Amino-6'-Deoxy-5'-Homothymidine, 42;6'-Amino-2',6'-Dideoxy-5'-Homoadenosine, 43;6'-Amino-2',6'-Dideoxy-5'-Homocytidine, 44; and6'-Amino-2',6'-Dideoxy-5'-Homoguanosine, 45 (Via An Intramolecular FreeRadical Reaction)

Deblocking of compound 10 is effected by treatment with Bu₄ NF in THF.The resulting compound 39 (also reported in Preparation example 4 ofEuropean Patent application 0 381 335 A1) will be iodinated upontreatment with methyltriphenoxyphosphonium iodide as per the procedureof Verheyden, J. P. H. and Moffatt, J. G., J. Org. Chem., 35:2119 (1970)to furnish 5'-deoxy-5'-iodo-3'-O-methyleneaminothymidine, compound 40.Compound 40 when subjected to an intramolecular free radical reactionaccording to the procedure of Curran, D. P., Radical Addition Reactions,In Comprehensive Organic Synthesis: Trost, B. M. and Fleming, I., Eds.,vol. 4, p 715-832, Pergamon Press, Oxford (1991), will give thecorresponding 3'-O-isoxazolidinethymidine, compound 41 which on DIBAL-Hreduction will yield 6'-amino-5'-homothymidine, compound 42 [the3'-(t-butyldimethylsilyl) derivative of this compound is reported inRawson, T. E. and Webb, T. R., Nucleosides & Nucleotides, 9:89 (1990)].

When reacted in a like manner compounds 27, 28 and 29 will give6'-amino-5'-homoadenosine, compound 43; 6'-amino-5'-homocytidine,compound 44; and 6'-amino-5'-homoguanosine, compound 45.

EXAMPLE 31 3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-C-Allylthymidine 46

A stirred solution of3'-O-(t-butyldiphenylsilyl)-5'-deoxy-5'-iododthymidine (12, 1.77 g, 3mmol), allytributyltin (2.97 g, 9 mmol) and AIBN (0.54 g, 3.3 mmol) indry toluene (30 ml) was degassed completely and heated at 65° C. for 6hr. The solution was cooled and concentrated under vacuo. The residuewas purified by silica gel column chromatography and on elution withhexanes:EtOAc (1:1, v/v) furnished the title compound as homogeneousmaterial. Appropriate fractions were pooled and evaporated to furnish46, 0.75 g of a white foam, 50% yield. The structure was confirmed by ¹H NMR.

EXAMPLE 32 3'-O-(t-Butyldiphenylsilyl)-5-Deoxy-7'-C-Aldehydothymidine 47

A solution of 46 (1 mmol), OsO₄ (0.1 mmol) and nmethylmorpholine oxide(2 mmol) in diethyl ether (4 ml) and water (2 ml) are stirred for 18 hrat room temperature. A solution of NaIO₄ (3 ml) is added and thesolution further stirred for 12 hr. The aqueous layer is extracted withdiethyl ether. Evaporation of the organic layer will give the crudealdehyde 47.

EXAMPLE 335'-O-(t-Butyldimethylsilyl)-3'-O-Dephosphinico-3'-O-(Iminomethylene)thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Hydroxythymidine,48

Utilizing the procedure of Hanamoto, T. and Inanaga, J., Tet. Letts.,32:3555 (1991), SmI₂ (0.1 mmol) in THF (3 ml) is added to a mixture ofcompound 5 and compound 10 in HMPA (0.5 ml) with stirring. The mixturewill be stirred at room temperature for about 15 mins to form the adduct(as detected by the fading color). The solvent will be removed and theresidue purified by column chromatography to give the dimericoligonucleoside 48.

EXAMPLE 343'-O-Dephosphinico-3'-O-[N-(Morpholin-2-yl)]thymidylyl-(3'→4')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-5'-Demethylenethymidine,49

Utilizing the modification of Lim, M.-I. and Pan, Y.-G., Book ofAbstracts, 203 ACS national Meeting, San Francisco, Calif.., Apr. 5-10,1992, of the procedure of Hill, J. and Ramage, G. R. J., J. Chem. Soc.,3709 (1964), the dimeric oligonucleoside of Example 33 (compound 48, 1equiv.) will be treated with chloroacetyl chloride in acetone to form anadduct with the amino group of the linkage. Further treatment with K₂CO₃ (1.2 equiv.) in DMSO at elevated temperature will cyclize the adductto the hydroxyl group of the linkage to form a 5-oxomorpholino adductwith the linkage. The oxomorpholino adduct is then reduced with BH₃ -THFunder reflux to yield the dimer linked via an--O--[N-(morpholin-2-yl)]-linkage, compound 49.

EXAMPLE 35N3-Benztoyl-1-(5'-O-Dimethoxytrityl-3'-O-Trifluoromethylsulfonyl-threo-Pentofuranosyl)thymine,50

The method of Horwitz, J. P. et al., J. Org. Chem., 29:2076 (1964) willbe utilized to prepare the title compound with substitution of thetrifluoromethanesulfonic anhydride/pyridine (-50° C. to 0° C.) reactionconditions of Fleet, G. W. J. et al., Tetrahedron, 44:625 (1988) for themethylsulfonic anhydride conditions of Horwitz et al.

EXAMPLE 36 6'-O-Phthalimido-5'-Homothymidine, 52

To a stirred mixture of 5'-homothymidine [Etzold, G., Kowollik, G., andLangen, R., Chemical Communications, pg 422 (1968)] (51, 1.28. g, 5mmol), N-hydroxyphthalimide (1.09 g, 6.6 mmol) and triphenylphosphine(1.75 g, 6.6 mmol) in dry DMF (25 ml) will be addeddiisopropylazodicarboxylate (1.5 ml, 7.5 mmol) over a period of 30 minat 0° C. The stirring is continued for 12 hr at room temperature. Thesolvent is evaporated under vacuo and the residue is washed with diethylether (2×50 ml). The residue will then be suspended in hot EtOH (50 ml),cooled and filtered to give the title compound 52.5

EXAMPLE 37 6'-O-Phthalimido-3'-O-(t-Butyldiphenylsilyl)-Homothymidine53

Compound 52 will be treated with t-butyldiphenylchlorosilane in pyridineand imidazole in a standard manner to afford the title compound 53.

EXAMPLE 38 6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine, 54

To a stirred solution of compound 53 in dry CH₂ Cl₂ is addedmethylhydrazine (3 mmol) under anhydrous conditions at room temperature.The solution is stirred for 12 hr, cooled (0° C.) and filtered. Theprecipitate will be washed with CH₂ Cl₂ and the combined filtrates willbe concentrated. The residue is purified by flash column chromatography(silica gel, 20 g). Elution with CH₂ Cl₂ :MeOH, 9:1, v/v) will furnishthe title compound 54.

EXAMPLE 393'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Tritylthymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,55

6'-O-Amino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine, 54, isconverted to the corresponding urethane with ethyl chloroformate (CH₂Cl₂ -saturated NaHCO₃) utilizing the stereospecific conditions of Yang,D., Kim, S.-H. and Kahne, D., J. Am. Chem. Soc., 113:4715 (1991). Theresidue of this reaction will then be stirred in CH₂ Cl₂ with compound50. The products are then concentrated in vacuo to yield the dimericoligonucleoside, compound 55.

EXAMPLE 403'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Tritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,56

Compound 55 will be N-alkylated as per the conditions of Step 3 ofExample 4 to yield the N-alkylate iminooxymethylene linked dimericoligonucleoside 56.

EXAMPLE 413'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-5'-Deoxythymidine,57

The 5'-O-trityl and the 3'-O-(t-butyldiphenylsilyl) protecting groups ofcompound 56 will be removed by treatment with trifluoroacetic acid andthe residue dimethoxytritylated as per the procedure of Sproat, B. S.and Lamond, A. I., 2'-O-Methyloligoribonucleotides: synthesis andapplications, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, pg. 55 (1991), to give the title compound.

EXAMPLE 423'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(diisopropyl)phosphiryl]-5'-Deoxythymidine,58

Compound 57 (1.89 mmol) will be dissolved in anhydrous dichloromethaneunder an argon atmosphere. Diisopropylethylamine (0.82 ml, 4.66 mmol) isadded and the reaction mixture cooled to ice temperature.Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 ml, 4.03 mmol) isadded to the reaction mixture and the reaction mixture is allowed towarm to 20° C. and stirred for 3 hr. Ethylacetate (80 ml) andtriethylamine (1 ml) are added and the solution is washed with brinesolution three times (3×25 ml). The organic phase is separated and driedover magnesium sulfate. After filtration of the solids the solvent isevaporated in vacuo at 20° C. to an oil that will then be purified bycolumn chromatography using silica and a solvent such as hexane-ethylacetatetriethylamine (50:40:1) as eluent. The fractions are thenevaporated in vacuo and the residue will be further evaporated withanhydrous pyridine (20 ml) in vacuo (1 torr) at 26° C. in the presenceof sodium hydroxide for 24 hr to yield the title compound 58.

EXAMPLE 43 5'-Amino-5'-Homothymidine, 60

5'-Amino-3'-O-(t-butyldimethylsilyl)-5'-homothymidine 59 is prepared asper Rawson, T. E., and Webb, T. R., Nucleosides & Nucleotides, 9:89(1990). The t-butyldimethylsilyl group will be removed as per theprocedure of Step 4 of Example 4 to give the title compound.

EXAMPLE 44 5'-Methylamino-3'-O-(t-Butyldiphenylsilyl)-5'-Homothymidine,62

Compound 60 is t-butyldiphenylsilated as per the procedure of 37 to give5'-Amino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine, compound 61,which will then be treated as per the procedure of Step 3 of Example 4alkylate the 5'-amino group to yield the title compound 62.

EXAMPLE 453'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,64

5'-Methylamino-3'-O-(t-butyldiphenylsilyl)-5'-homothymidine 62 (1 mmol)will be added to aqueous sodium hypochloride (4 mmol) to furnish achloramide intermediate. The chloramide intermediate is cooled (0° C.)and treated with 5'-O-monomethoxytrity-3'-thiothymidine (0.9 mmol ) ,compound 63, prepared as per Cosstick, R. and Vyle, J. S., Nucleic AcidsRes., 18: 829 (1990) . The reaction mixture is worked up utilizing theprocedure of Barton, D. H. R. et al., J. Org. Chem., 56:6702 (1991) andthe residue will be purified by chromatography to give the titlecompound 64.

EXAMPLE 463'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-5'-Deoxythymidine,65

Compound 64 will be deblocked at the terminal 3' position utilizing theas per the procedure of Step 4 of Example 4 to give compound 65.

EXAMPLE 473'-Dephosphinico-3'-S-[(Methylimino)methylene]-5'-Monomethoxytrityl-3'-Thiothymidylyl-(3'→5')-3'[(β-Cyanoethoxy)-N-(diisopropyl)phosphortityl]-5'-Deoxythymidine66

Compound 65 will be phosphitylated as per the procedure of Example 42 togive the title compound 66.

EXAMPLE 485'-O-(t-Butyldimethylsilyl)-3'-De(oxyphosphinico)-3'-(Imino-1,2-Ethanediyl)thymidylyl-(3'→5')3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,68

3'-Amino'-5'-O-(t-butyldimethylsilyl)-3'-deoxythymidine, compound 67,prepared according to Matsuda, A., Satoh, M. and Ueda, T., Nucleoside &Nucleotides, 9:587 (1990) will be reductively coupled with compound 47in the presence of a catalytic amount of acid as per the procedure ofMagid et. al, Tett. Lets., 31:5595 (1990), to yield the Schiff's baseintermediate that is reduced in situ to give the amino linkage of thetitle compound 68.

EXAMPLE 493'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'.fwdarw.5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,69

Compound 68 will be methylated and deblocked at the 5' position as perthe procedure of Step 3 of Example 4 to yield the N-alkylated5'-deblocked dimer, compound 69.

EXAMPLE 503'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,70

Compound 69 will be dimethoxytritylated as per the procedure of Sproat,B. S. and Lamond, A. I., 2'-O-Methyloligoribonucleotides: synthesis andapplications, Oligonucleotides and Analogs A Practical Approach, F.Eckstein Ed., IRL Press, pg. 55 (1991).

EXAMPLE 513'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-5'-Deoxythymidine,71

The dimethoxytritylated intermediate, compound 70 when deblocked at the3' terminus as per the procedure of Step 4 of Example 4 will givecompound 71.

EXAMPLE 523'-De(oxyphosphinico)-5'-Dimethoxytrityl-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-[(S-Cyanoethoxy)-N-(diisopropyl)phosphiryl]-5'-Deoxythymidine,72

Compound 71 will be phosphitylated as per the procedure of Example 42 togive the title compound 72.

EXAMPLE 53 2'-O-Methylhomoadenosine, 74

Homoadenosine, 73, prepared as per the procedure of Kappler, F. andHampton, A., Nucleic Acid Chemistry, Part 4, Ed. L. B. Townsend and R.S. Tipson, Wiley-Interscience Publication, pg. 240 (1991), will beblocked across its 3' and 5' hydroxyl groups with a TIPS, i.e.tetraisopropylsilyl, blocking group followed by alkylation as per theprocedures described in United States patent applications 566,977, filedAug. 13, 1990 and PCT/US91/05720, filed Aug. 12, 1991. Removal of theTIPS group with tetra-n-butylammonium fluoride as per the procedure ofStep 4 of Example 4 will yield the title compound 74.

EXAMPLE 54 6'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-5'-Homoadenosine, 75

Compound 74 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 75.

EXAMPLE 553'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxy-2'-O-Methyladenosine,76

Compound 75 will be treated and reacted with compound 50 as per theprocedure of Example 39 to yield the title compound 76.

EXAMPLE 563'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(S-Cyanoethoxy)-N-(Diisopropyl)phosphiryl]-5'-Deoxy-2'-O-Methyladenosine,77

Compound 76 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 77.

EXAMPLE 576'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-Deoxy-5'-Homoaristeromycin, 79

(-)-2'-Deoxy-5'-homoaristeromycin, compound 78, (the carbocyclicanalogue of 5'-homo-2'-deoxyadenosine) is prepared as per the procedureof Jones, M. F. and Roberts, S. M., J. Chem. Soc. Perkin Trans., 1:2927(1988). Compound 78 will be treated as per the procedure of Examples 36,37 and 38 to yield the 6'-O-amino-3'-blocked carbocyclic analogue of5'-homo-2'-deoxyadenosine, compound 79.

EXAMPLE 583'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-2',5'-Dideoxyaristeromycin,80

Compound 79 will be treated and reacted with compound 50 as per theprocedure of Example 39 to yield the title compound 80.

EXAMPLE 593'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl]-2',5'-Dideoxyaristeromycin,81

Compound 80 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 81.

EXAMPLE 60 6'-O-Amino-2'-O-Butyl-5'-Homoaristeromycin, 82

(-)-5'-Homoaristeromycin, compound 78, will be blocked with a TIPSgroup, alkylated and deblocked as per the procedure of Example 57 toyield compound 82.

EXAMPLE 616'-O-Amino-3'-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Homoaristeromycin,83

Compound 82 will be treated as per the procedures of Examples 36, 37 and38 to yield the title compound 83.

EXAMPLE 623'-De(oxophosphinico)-3'-(Iminooxymethylene)-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-2'-O-Butyl-5'-Deoxyaristeromycin,84

Compound 83 will be treated and reacted with compound 50 as per theprocedure of Example 39 to yield the title compound 84.

EXAMPLE 633'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-5'-Dimethoxytritylthymidylyl-(3'→5')-3'-[(β-Cyanoethoxy)-N-(Diisopropyl)phosphiryl]-2'-O-Butyl-5'-Deoxyaristeromycin,85

Compound 84 will be reacted as per the reaction sequence of Examples 40,41 and 42 to yield the title compound 85.

EXAMPLE 64(+)-1-[(1R,3S,4S)-3-Azido-5-Dimethoxytrityl-4-(Hydroxymethyl)-Cyclopentyl]-5-Methyl-2,4-(1H,3H)-Pyrimidindione,87

(+)-1-[1R,3S,4S)-3-Azido-4-(hydroxymethyl)-cyclopentyl]-5-methyl-2,4-(1H,3H)-pyrimidindione,compound 86, prepared as per the procedure of Bodenteich, M. and Grieng,H., Tetrahedron Letts., 28:5311 (1987), will be dimethoxytritylatedutilizing dimethoxytrityl chloride in pyridine at room temperature togive the title compound 87.

EXAMPLE 65(+)-1-[(1R,3S,4S)-3-Amino-4-(Dimethoxytrityloxymethyl)-Cyclopentyl]-5-Methyl-2,4-(1H,3H)-Pyrimidindione,88

Compound 87 will be reduced with Ph₃ P in pyridine at room temperatureas per the procedure of Hronowski, L. J. J. and Szarek, W. A., J. Chem.Soc., Chem. Commun., 1547 (1990), to give the carbocyclic analogue of3'-amino-5'-dimethoxytrityl thymidine, compound 88.

EXAMPLE 661-{(1R,3S,4S)-3-[Imino-2-(5'-Deoxythymidylyl-5'-yl)-1,2-Ethanediyl]-4-(Dimethoxtrityloxymethyl)-Cyclopentyl}-5-Methyl-2,4-(1H,3H)-Pyrimidindione,89

Compound 88 will be reacted with compound 47 as per the procedure ofExample 48 to yield the title compound 89.

EXAMPLE 67 Synthesis Of Oligonucleotides Using A DNA Synthesizer

Solid support oligonucleotide and "oligonucleotide like" syntheses areperformed on an Applied Biosystems 380 B or 394 DNA synthesizerfollowing standard phosphoramidite protocols and cycles using reagentssupplied by the manufacture. The oligonucleotides are normallysynthesized in either a 10 μmol scale or a 3×1 μmol scale in the"Trityl-On" mode. Standard deprotection conditions (30% NH₄ OH, 55° C.,16 hr) are employed. HPLC is performed on a Waters 600E instrumentequipped with a model 991 detector. For analytical chromatography, thefollowing reverse phase HPLC conditions are employed: Hamilton PRP-1column (15×2.5 cm); solvent A: 50 mm TEAA, pH 7.0; solvent B: 45 mm TEAAwith 80% CH₃ CN; flow rate: 1.5 ml/min; gradient: 5% B for the first 5minutes, linear (1%) increase in B every minute thereafter. Forpreparative purposes, the following reverse phase HPLC conditions areemployed: Waters Delta Pak Waters Delta-Pak C₄ 15 μm, 300A, 25×100 mmcolumn equipped with a guard column of the same material; column flowrate: 5 ml/min; gradient: 5% B for the first 10 minutes, linear 1%increase for every minute thereafter. Following HPLC purification,oligonucleotides are detritylated and further purified by size exclusionusing a Sephadex G-25 column.

EXAMPLE 68 HIGHER ORDER MIXED OLIGONUCLEOSIDES-OLIGONUCLEOSIDES ANDMIXED OLIGONUCLEOSIDES-OLIGONUCLEOTIDES

A. Solution Phase Synthesis Of3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-3'-Phosphorothioate-Thymidylyl-(3'→5')-3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl]thymidylyl-(3'→5')-3'-O-(t-Butyldiphenylsilyl)-5'-Deoxythymidine,90, A Mixed Oligonucleoside-Oligonucleotide-Oligonucleoside PolymerIncorporating A Nucleotide Linkage Flanked At Its 5' Terminus By A3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)] LinkedOligonucleoside Dimer and At Its 3' Terminus By A3'-De(oxyphosphinico)-3'-[(Methylimino)-1,2-Ethanediyl] LinkedOligonucleoside Dimer

A mixed oligonucleoside-oligonucleotide-oligonucleoside having a3'-de(oxophosphinico)-3'-[methyl(iminooxymethylene)] linkedoligonucleoside dimer and a3'-de(oxyphosphinico)-3'-[(methylimino)-1,2-ethanediyl] linkedoligonucleoside dimer coupled together via a phosphorothioate nucleotidelinkage will be prepared by reacting compound 58, compound 70 andtetrazole in anhydrous acetonitrile under argon. The coupling reactionwill be allowed to proceed to completion followed by treatment withBeaucage reagent and ammonium hydroxide removal of the dimethoxytritylblocking group according to the procedure of Zon, G. and Stec, W. J.,Phosphorothioate oligonucleotides, Oligonucleotides and Analogs APractical Approach, F. Eckstein Ed., IRL Press, pg. 87 (1991). The 3'blocking group will then removed as per the procedure of Step 3 ofExample 4 and the product purified by HPLC to yield the title compound90, wherein utilizing the structure of Scheme XVIII, T₃ and T₅ are OH, Dis S, E is OH, X is H, Q is O, r is 0 and q is 2; and for each q, i.e.q₁ and q₂, n and p are 1 in each instance; and for q₁, m is 1; and forq₂, m is 0; and Bxj and Bxi are thymine.

B. Solid Support Synthesis Of3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-Thymidylyl-(3'.fwdarw.5')-5'-Deoxythymidylyl-(3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-(3'→5')-Thymidylyl-(3'→5')-P-Thymidylyl-3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)]-(3'→5')-Thymidylyl-(3'→5')-P-2'-Deoxycytidine,91, A Mixed Oligonucleotide-Oligonucleoside Polymer Incorporating3'-De(oxophosphinico)-3'-[Methyl(iminooxymethylene)] LinkedOligonucleoside Dimers Flanked By Conventional Linked Nucleotides

The dimeric oligonucleoside 58 will be utilized as building block unitsin a conventional oligonucleotide solid support synthesis as per theprocedure of Example 67. For the purpose of illustration a polymerincorporating seven nucleosides is described. A first unit of thedimeric oligonucleoside 58 will be coupled to a first cytidinenucleoside tethered to a solid support via its 3' hydroxyl group andhaving a free 5' hydroxyl group. After attachment of the first unit ofcompound 58 to the support, the 5'-dimethoxytrityl group of that firstcompound 58 unit will be removed in the normal manner. A second compound58 unit will then be coupled via itsβ-cyanoethyl-N-diisopropylphosphiryl group to the first compound 58 unitusing normal phosphoramidate chemistry. This forms a conventionalphosphodiester bond between the first and second compound 58 units andelongates the polymer by two nucleosides (or one oligonucleoside dimerunit). The dimethoxytrityl blocking group from the second compound 58unit will be removed in the normal manner and the polymer elongated by afurther dimeric unit of compound 58. As with addition of the first andsecond dimeric units, the third unit of compound 58 is coupled to thesecond via conventional phosphoramidite procedures. The addition of thethird unit of compound 58 completes the desired length and basesequence. This polymer has a backbone of alternating normalphosphodiester linkages and the methyl(iminooxymethylene) linkages ofcompound 58. The 5' terminal dimethoxytrityl group of the third compound58 unit will be removed in the normal manner followed by release of thepolymer from the solid support, also in the normal manner. Purificationof the polymer will be achieved by HPLC to yield compound 91 wherein,utilizing the structure of Scheme XVIII, T₃ and T₅ are OH, D is O, E isOH, X is H, Q is O, r is 1 and for the seven nucleoside polymerdescribed, q is 3; and for each q, i.e. q₁, q₂ and q₃, n and p are 1 ineach instances; and for q₁ and q₂, m is 1; and for q₃, m is 0; and Bxkis cytosine; and each BxJ and Bxi is thymine.

EVALUATION

PROCEDURE 1--Nuclease Resistance

A. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to serum and cytoplasmic nucleases.

Oligonucleotide-mimicking macromolecules of the invention can beassessed for their resistance to serum nucleases by incubation of theoligonucleotide-mimicking macromolecules in media containing variousconcentrations of fetal calf serum or adult human serum. Labeledoligonucleotide-mimicking macromolecules are incubated for varioustimes, treated with protease K and then analyzed by gel electrophoresison 20% polyacrylamine-urea denaturing gels and subsequentautoradiography. Autoradiograms are quantitated by laser densitometry.Based upon the location of the modified linkage and the known length ofthe oligonucleotide-mimicking macromolecules it is possible to determinethe effect on nuclease degradation by the particular modification. Forthe cytoplasmic nucleases, an HL 60 cell line can be used. Apost-mitochondrial supernatant is prepared by differentialcentrifugation and the labelled macromolecules are incubated in thissupernatant for various times. Following the incubation, macromoleculesare assessed for degradation as outlined above for serum nucleolyticdegradation. Autoradiography results are quantitated for evaluation ofthe macromolecules of the invention. It is expected that themacromolecules will be completely resistant to serum and cytoplasmicnucleases.

B. Evaluation of the resistance of oligonucleotide-mimickingmacromolecules to specific endo- and exo-nucleases.

Evaluation of the resistance of natural oligonucleotides andoligonucleotide-mimicking macromolecules of the invention to specificnucleases (ie, endonucleases, 3',5'-exo-, and 5',3'-exonucleases) can bedone to determine the exact effect of the macromolecule linkage ondegradation. The oligonucleotide-mimicking macromolecules are incubatedin defined reaction buffers specific for various selected nucleases.Following treatment of the products with protease K, urea is added andanalysis on 20% polyacrylamide gels containing urea is done. Gelproducts are visualized by staining with Stains All reagent (SigmaChemical Co.). Laser densitometry is used to quantitate the extent ofdegradation. The effects of the macromolecules linkage are determinedfor specific nucleases and compared with the results obtained from theserum and cytoplasmic systems. As with the serum and cytoplasmicnucleases, it is expected that the oligonucleotide-mimickingmacromolecules of the invention will be completely resistant to endo-and exo-nucleases.

PROCEDURE 2--5-Lipoxygenase Analysis and Assays

A. Therapeutics

For therapeutic use, an animal suspected of having a diseasecharacterized by excessive or abnormal supply of 5-lipoxygenase istreated by administering the macromolecule of the invention. Persons ofordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Such treatment is generallycontinued until either a cure is effected or a diminution in thediseased state is achieved. Long term treatment is likely for somediseases.

B. Research Reagents

The oligonucleotide-mimicking macromolecules of this invention will alsobe useful as research reagents when used to cleave or otherwise modulate5-lipoxygenase mRNA in crude cell lysates or in partially purified orwholly purified RNA preparations. This application of the invention isaccomplished, for example, by lysing cells by standard methods,optimally extracting the RNA and then treating it with a composition atconcentrations ranging, for instance, from about 100 to about 500 ng per10 Mg of total RNA in a buffer consisting, for example, of 50 mmphosphate, pH ranging from about 4-10 at a temperature from about 30° toabout 50° C. The cleaved 5-lipoxygenase RNA can be analyzed by agarosegel electrophoresis and hybridization with radiolabeled DNA probes or byother standard methods.

C. Diagnostics

The oligonucleotide-mimicking macromolecules of the invention will alsobe useful in diagnostic applications, particularly for the determinationof the expression of specific mRNA species in various tissues or theexpression of abnormal or mutant RNA species. In this example, while themacromolecules target a abnormal mRNA by being designed complementary tothe abnormal sequence, they would not hybridize to normal mRNA.

Tissue samples can be homogenized, and RNA extracted by standardmethods. The crude homogenate or extract can be treated for example toeffect cleavage of the target RNA. The product can then be hybridized toa solid support which contains a bound oligonucleotide complementary toa region on the 5' side of the cleavage site. Both the normal andabnormal 5' region of the mRNA would bind to the solid support. The 3'region of the abnormal RNA, which is cleaved, would not be bound to thesupport and therefore would be separated from the normal mRNA.

Targeted mRNA species for modulation relates to 5-lipoxygenase; however,persons of ordinary skill in the art will appreciate that the presentinvention is not so limited and it is generally applicable. Theinhibition or modulation of production of the enzyme 5-lipoxygenase isexpected to have significant therapeutic benefits in the treatment ofdisease. In order to assess the effectiveness of the compositions, anassay or series of assays is required.

D. In Vitro Assays

The cellular assays for 5-lipoxygenase preferably use the humanpromyelocytic leukemia cell line HL-60. These cells can be induced todifferentiate into either a monocyte like cell or neutrophil like cellby various known agents. Treatment of the cells with 1.3% dimethylsulfoxide, DMSO, is known to promote differentiation of the cells intoneutrophils. It has now been found that basal HL-60 cells do notsynthesize detectable levels of 5-lipoxygenase protein or secreteleukotrienes (a downstream product of 5-lipoxygenase). Differentiationof the cells with DMSO causes an appearance of 5-lipoxygenase proteinand leukotriene biosynthesis 48 hours after addition of DMSO. Thusinduction of 5-lipoxygenase protein synthesis can be utilized as a testsystem for analysis of oligonucleotide-mimicking macromolecules whichinterfere with 5-lipoxygenase synthesis in these cells.

A second test system for oligonucleotide-mimicking macromolecules makesuse of the fact that 5-lipoxygenase is a "suicide" enzyme in that itinactivates itself upon reacting with substrate. Treatment ofdifferentiated HL-60 or other cells expressing 5 lipoxygenase, with 10μM A23187, a calcium ionophore, promotes translocation of 5-lipoxygenasefrom the cytosol to the membrane with subsequent activation of theenzyme. Following activation and several rounds of catalysis, the enzymebecomes catalytically inactive. Thus, treatment of the cells withcalcium ionophore inactivates endogenous 5-lipoxygenase. It takes thecells approximately 24 hours to recover from A23187 treatment asmeasured by their ability to synthesize leukotriene B₄. Macromoleculesdirected against 5-lipoxygenase can be tested for activity in two HL-60model systems using the following quantitative assays. The assays aredescribed from the most direct measurement of inhibition of5-lipoxygenase protein synthesis in intact cells to more downstreamevents such as measurement of 5-lipoxygenase activity in intact cells.

A direct effect which oligonucleotide-mimicking macromolecules can exerton intact cells and which can be easily be quantitated is specificinhibition of 5-lipoxygenase protein synthesis. To perform thistechnique, cells can be labelled with ³⁵ S-methionine (50 μCi/mL) for 2hours at 37° C. to label newly synthesized protein. Cells are extractedto solubilize total cellular proteins and 5-lipoxygenase isimmunoprecipitated with 5-lipoxygenase antibody followed by elution fromprotein A Sepharose beads. The immunoprecipitated proteins are resolvedby SDS-polyacrylamide gel electrophoresis and exposed forautoradiography. The amount of immunoprecipitated 5-lipoxygenase isquantitated by scanning densitometry.

A predicted result from these experiments would be as follows. Theamount of 5-lipoxygenase protein immunoprecipitated from control cellswould be normalized to 100%. Treatment of the cells with 1 μM, 10 μM,and 30 μM of the macromolecules of the invention for 48 hours wouldreduce immunoprecipitated 5-lipoxygenase by 5% 25% and 75% of control,respectively.

Measurement of 5-lipoxygenase enzyme activity in cellular homogenatescould also be used to quantitate the amount of enzyme present which iscapable of synthesizing leukotrienes. A radiometric assay has now beendeveloped for quantitating 5-lipoxygenase enzyme activity in cellhomogenates using reverse phase HPLC. Cells are broken by sonication ina buffer containing protease inhibitors and EDTA. The cell homogenate iscentrifuged at 10,000×g for 30 min and the supernatants analyzed for5-lipoxygenase activity. Cytosolic proteins are incubated with 10 μM ¹⁴C-arachidonic acid, 2 mM ATP, 50 μM free calcium, 100 μg/mlphosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 37°C. The reactions are quenched by the addition of an equal volume ofacetone and the fatty acids extracted with ethyl acetate. The substrateand reaction products are separated by reverse phase HPLC on a NovapakC18 column (Waters Inc., Millford, Mass.). Radioactive peaks aredetected by a Beckman model 171 radiochromatography detector. The amountof arachidonic acid converted into di-HETE's and mono-HETE's is used asa measure of 5-lipoxygenase activity.

A predicted result for treatment of DMSO differentiated HL-60 cells for72 hours with effective the macromolecules of the invention at 1 μM, 10μM, and 30 μM would be as follows. Control cells oxidize 200 pmolarachidonic acid/5 min/10⁶ cells. Cells treated with 1 μM, 10 μM, and 30μM of an effective oligonucleotide-mimicking macromolecule would oxidize195 pmol, 140 pmol, and 60 pmol of arachidonic acid/5 min/10⁶ cellsrespectively.

A quantitative competitive enzyme linked immunosorbant assay (ELISA) forthe measurement of total 5-lipoxygenase protein in cells has beendeveloped. Human 5-lipoxygenase expressed in E. coli and purified byextraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is usedas a standard and as the primary antigen to coat microtiter plates. 25ng of purified 5-lipoxygenase is bound to the microtiter platesovernight at 4° C. The wells are blocked for 90 min with 5% goat serumdiluted in 20 mM Tris•HCL buffer, pH 7.4, in the presence of 150 mM NaCl(TBS). Cell extracts (0.2% Triton X-100, 12,000×g for 30 min.) orpurified 5-lipoxygenase were incubated with a 1:4000 dilution of5-lipoxygenase polyclonal antibody in a total volume of 100 μL in themicrotiter wells for 90 min. The antibodies are prepared by immunizingrabbits with purified human recombinant 5-lipoxygenase. The wells arewashed with TBS containing 0.05% tween 20 (TBST), then incubated with100 μL of a 1:1000 dilution of peroxidase conjugated goat anti-rabbitIgG (Cappel Laboratories, Malvern, Pa.) for 60 min at 25° C. The wellsare washed with TBST and the amount of peroxidase labelled secondantibody determined by development with tetramethylbenzidine.

Predicted results from such an assay using a 30 meroligonucleotide-mimicking macromolecule at 1 μM, 10 μM, and 30 μM wouldbe 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10⁶ cells, respectivelywith untreated cells containing about 34 ng 5-lipoxygenase.

A net effect of inhibition of 5-lipoxygenase biosynthesis is adiminution in the quantities of leukotrienes released from stimulatedcells. DMSO-differentiated HL-60 cells release leukotriene B4 uponstimulation with the calcium ionophore A23187. Leukotriene B4 releasedinto the cell medium can be quantitated by radioimmunoassay usingcommercially available diagnostic kits (New England Nuclear, Boston,Mass.). Leukotriene B4 production can be detected in HL-60 cells 48hours following addition of DMSO to differentiate the cells into aneutrophil-like cell. Cells (2×10⁵ cells/mL) will be treated withincreasing concentrations of the macromolecule for 48-72 hours in thepresence of 1.3% DMSO. The cells are washed and resuspended at aconcentration of 2×10⁶ cell/mL in Dulbecco's phosphate buffered salinecontaining 1% delipidated bovine serum albumin. Cells are stimulatedwith 10 μM calcium ionophore A23187 for 15 min and the quantity of LTB4produced from 5×10⁵ cell determined by radioimmunoassay as described bythe manufacturer.

Using this assay the following results would likely be obtained with anoligonucleotide-mimicking macromolecule directed to the 5-LO mRNA. Cellswill be treated for 72 hours with either 1 μM, 10 μM or 30 μM of themacromolecule in the presence of 1.3% DMSO. The quantity of LTB₄produced from 5×10⁵ cells would be expected to be about 75 pg, 50 pg,and 35 pg, respectively with untreated differentiated cells producing 75pg LTB₄.

E. In Vivo Assay

Inhibition of the production of 5-lipoxygenase in the mouse can bedemonstrated in accordance with the following protocol. Topicalapplication of arachidonic acid results in the rapid production ofleukotriene B₄, leukotriene C₄ and prostaglandin E₂ in the skin followedby edema and cellular infiltration. Certain inhibitors of 5-lipoxygenasehave been known to exhibit activity in this assay. For the assay, 2 mgof arachidonic acid is applied to a mouse ear with the contralateral earserving as a control. The polymorphonuclear cell infiltrate is assayedby myeloperoxidase activity in homogenates taken from a biopsy 1 hourfollowing the administration of arachidonic acid. The edematous responseis quantitated by measurement of ear thickness and wet weight of a punchbiopsy. Measurement of leukotriene B₄ produced in biopsy specimens isperformed as a direct measurement of 5-lipoxygenase activity in thetissue. Oligonucleotide-mimicking macromolecules will be appliedtopically to both ears 12 to 24 hours prior to administration ofarachidonic acid to allow optimal activity of the compounds. Both earsare pretreated for 24 hours with either 0.1 μmol, 0.3 μmol, or 1.0 μmolof the macromolecule prior to challenge with arachidonic acid. Valuesare expressed as the mean for three animals per concentration.Inhibition of polymorphonuclear cell infiltration for 0.1 μmol, 0.3μmol, and 1 μmol is expected to be about 10%, 75% and 92% of controlactivity, respectively. Inhibition of edema is expected to be about 3%,58% and 90%, respectively while inhibition of leukotriene B₄ productionwould be expected to be about 15%, 79% and 99%, respectively.

We claim:
 1. A nucleoside of the structure: ##STR4## wherein Y₁ is CH₂;Y₂ is aminooxy, alkylamino having 1 carbon atom, or alkenyl having 2carbon atoms; Z is H, OH, O--R'", amino, methyleneamino or phthalimido;R'" is a hydroxyl blocking group; X is H or OH; Q is O; and Bx is aheterocyclic base moiety.
 2. The nucleoside of claim 1 wherein Y₂ isaminooxy.
 3. The nucleoside of claim 1 wherein Y₂ is alkylamino having 1carbon atom.
 4. The nucleoside of claim 1 wherein Y₂ is alkenyl having 2carbon atoms.
 5. The nucleoside of claim 1 wherein Z is OH.
 6. Thenucleoside of claim 1 wherein Z is amino.
 7. The nucleoside of claim 1wherein Z is methyleneamino.
 8. The nucleoside of claim 1 wherein Z isphthalimido.